CA3034810C - In-situ solvent recycling process for solid phase peptide synthesis at elevated temperatures - Google Patents
In-situ solvent recycling process for solid phase peptide synthesis at elevated temperatures Download PDFInfo
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- CA3034810C CA3034810C CA3034810A CA3034810A CA3034810C CA 3034810 C CA3034810 C CA 3034810C CA 3034810 A CA3034810 A CA 3034810A CA 3034810 A CA3034810 A CA 3034810A CA 3034810 C CA3034810 C CA 3034810C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
- C07K1/045—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
- C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
An improvement in of deprotection in solid phase peptide synthesis is disclosed. The method includes the steps of adding the deprotection composition in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling cycle; and without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle; and with the coupling solution at a temperature of at least 30°C.
Description
IN-SITU SOLVENT RECYCLING PROCESS FOR
SOLID PHASE PEPTIDE SYNTHESIS AT ELEVATED TEMPERATURES
Related Applications [001] This application is a continuation in part of US Publication Application No.
2017/0226152, filed October 21, 2016, for "Improvements in Solid Phase Peptide Synthesis."
Background
SOLID PHASE PEPTIDE SYNTHESIS AT ELEVATED TEMPERATURES
Related Applications [001] This application is a continuation in part of US Publication Application No.
2017/0226152, filed October 21, 2016, for "Improvements in Solid Phase Peptide Synthesis."
Background
[002] Bruce Merrifield's pioneering development of solid phase peptide synthesis created a useful process for synthesis peptide chains through its use of filtration to remove reagents between steps. The process has involved repetitive cycles which include coupling and deprotection with washing and filtration in-between each step (Figure 1).
It has commonly been assumed that washing is required between each step to completely remove the reagents previously used so that they don't undesirably participate in the next step. This typically involves "insertions" which refer to the incorporation of an extra amino acid. This is thought to occur through either residual base removing the protecting group (Fmoc) on an amino acid recently coupled thereby allowing a second amino acid to "insert"; or through residual activated amino acid left behind during the subsequent deprotection step which could couple to deblocked sites thereby "inserting' an extra amino acid from the previous step. It was recently shown, however, that washing after the coupling step was not required for the successful synthesis of peptides. In this work the coupling step was drained and the deprotection solution was subsequently added to the vessel (J. Collins, K. Porter, S. Singh and G.
Vanier, "High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS)," Org. Lett., vol.
16, pp. 940-943, 2014) (Figure 2).
Summary
It has commonly been assumed that washing is required between each step to completely remove the reagents previously used so that they don't undesirably participate in the next step. This typically involves "insertions" which refer to the incorporation of an extra amino acid. This is thought to occur through either residual base removing the protecting group (Fmoc) on an amino acid recently coupled thereby allowing a second amino acid to "insert"; or through residual activated amino acid left behind during the subsequent deprotection step which could couple to deblocked sites thereby "inserting' an extra amino acid from the previous step. It was recently shown, however, that washing after the coupling step was not required for the successful synthesis of peptides. In this work the coupling step was drained and the deprotection solution was subsequently added to the vessel (J. Collins, K. Porter, S. Singh and G.
Vanier, "High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS)," Org. Lett., vol.
16, pp. 940-943, 2014) (Figure 2).
Summary
[003] The invention is a method of deprotection in solid phase peptide synthesis in which the improvement comprises adding the deprotection composition in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling cycle;
and without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle; and with the coupling solution at least 30 C.
and without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle; and with the coupling solution at least 30 C.
[004] In another aspect the invention is a method of deprotection in solid phase peptide synthesis in which the improvement comprises adding the deprotection Date Recue/Date Received 2022-07-06 composition in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling cycle; and without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle which removes at least 50% of the volume of the previous cycle coupling solution;
and with the coupling solution at a temperature of at least 30 C.
[004a1 In another aspect, a method of deprotection in batch-wise solid phase peptide synthesis comprising: adding a base deprotection composition to a mixture of a coupling solution, a growing peptide chain, and any excess activated amino acid from a preceding coupling step; wherein the deprotection composition is added to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling step in an amount that is less than 1/3 of the volume of the coupling solution; without any draining step between the coupling step of the preceding coupling step and the addition of the deprotection composition for a successive cycle; and with the coupling solution at a temperature of at least 30 C.
[0041)1 In yet another aspect, a method of deprotection in batch-wise solid phase peptide synthesis comprising: adding a base deprotection composition to a mixture of a coupling solution, a growing peptide chain, and any excess activated amino acid from a preceding coupling cycle step; wherein the deprotection composition is added to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling cycle step in an amount that is less than 1/3 of the volume of the coupling solution; without any draining step between the coupling step of the preceding coupling cycle step and the addition of the deprotection composition for a successive cycle which removes at least 50% of the volume of the preceding coupling cycle step coupling solution; and with the coupling solution at a temperature of at least 30 C.
[0051 The foregoing and other objects of aspects and advantages of the invention and the manner in which the same are accomplished will become clearer based on the followed detailed description taken in conjunction with the accompanying drawings.
Brief Description of the Drawings [006] Figure 1 illustrates a traditional SPPS Cycle [007] Figure 2 illustrates more recent SPPS Cycles for High Efficiency Solid Phase Peptide Synthesis (HE-SPPS) Date Recue/Date Received 2022-07-06 [008] Figure 3 illustrates in-situ solvent recycling process for solid phase peptide synthesis.
Detailed Description [009] This invention presents a novel process whereby the coupling and deprotection steps occur within the same solvent. In this process concentrated base is added directly to the resin coupling solution after a desired period of time for the coupling to occur. The deprotection step is then immediately started when the base is added.
Therefore, the onset of the deprotection step is immediately after the coupling step without any time delay.
[010] Additionally, only a small volume of base is required since it can use the solvent present from the coupling reaction. This requires a sophisticated reagent delivery system for the base that is accurate at very small volumes (0.5mL) with rapid delivery.
Typically, a 20% solution of base (piperidine) hi_ solvent is used for the deprotection step. Excess base concentration can increase base-catalyzed side reactions and therefore significant solvent is required. This means that significant solvent can be saved from this process by adding concentrated base to the coupling solvent.
[011] To demonstrate the effectiveness of this new process a batch of 24 peptides were assembled using an automated peptide synthesizer modified to perform the in-situ solvent recycling step during each cycle.
[012] Materials and methods:
2a Date Recue/Date Received 2022-07-06 [013] All peptides were synthesized using a LTBERTY BLUETM PRTMETm system (CEM Corp., Matthews, NC, USA) allowing for automated in-situ solvent recycling and evaporation based washing. The peptides were synthesized at 0.05mmol scale with 10 equivalents of amino acid using CarbolVIAX1 m coupling with amino acidicarbodiimide/
ethyl 2-cyano-2-(hydroxyimino)acetate (AA/DIC/Oxyma) (1:2:1) based activation for 100 sec at 90 C E. Atherton, N. L. Benoiton, E. Brown, R. Sheppard and B. J.
Williams, "Racemization of Activated, Urethane-protected Amino-acids by p-Dimethylaminopyridine. Significance in Solid Phase Peptide Synthesis," J.C.S
(Ihem.
Comm., pp. 336-337, 1981). ProTide resins (CEM Corp.) based on TentaGele technology were used for synthesis with either a Rink Amide linker or a Cl-TCP(C1) linker with unactivated loading of the first amino acid with DIEA at 90 C for
and with the coupling solution at a temperature of at least 30 C.
[004a1 In another aspect, a method of deprotection in batch-wise solid phase peptide synthesis comprising: adding a base deprotection composition to a mixture of a coupling solution, a growing peptide chain, and any excess activated amino acid from a preceding coupling step; wherein the deprotection composition is added to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling step in an amount that is less than 1/3 of the volume of the coupling solution; without any draining step between the coupling step of the preceding coupling step and the addition of the deprotection composition for a successive cycle; and with the coupling solution at a temperature of at least 30 C.
[0041)1 In yet another aspect, a method of deprotection in batch-wise solid phase peptide synthesis comprising: adding a base deprotection composition to a mixture of a coupling solution, a growing peptide chain, and any excess activated amino acid from a preceding coupling cycle step; wherein the deprotection composition is added to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling cycle step in an amount that is less than 1/3 of the volume of the coupling solution; without any draining step between the coupling step of the preceding coupling cycle step and the addition of the deprotection composition for a successive cycle which removes at least 50% of the volume of the preceding coupling cycle step coupling solution; and with the coupling solution at a temperature of at least 30 C.
[0051 The foregoing and other objects of aspects and advantages of the invention and the manner in which the same are accomplished will become clearer based on the followed detailed description taken in conjunction with the accompanying drawings.
Brief Description of the Drawings [006] Figure 1 illustrates a traditional SPPS Cycle [007] Figure 2 illustrates more recent SPPS Cycles for High Efficiency Solid Phase Peptide Synthesis (HE-SPPS) Date Recue/Date Received 2022-07-06 [008] Figure 3 illustrates in-situ solvent recycling process for solid phase peptide synthesis.
Detailed Description [009] This invention presents a novel process whereby the coupling and deprotection steps occur within the same solvent. In this process concentrated base is added directly to the resin coupling solution after a desired period of time for the coupling to occur. The deprotection step is then immediately started when the base is added.
Therefore, the onset of the deprotection step is immediately after the coupling step without any time delay.
[010] Additionally, only a small volume of base is required since it can use the solvent present from the coupling reaction. This requires a sophisticated reagent delivery system for the base that is accurate at very small volumes (0.5mL) with rapid delivery.
Typically, a 20% solution of base (piperidine) hi_ solvent is used for the deprotection step. Excess base concentration can increase base-catalyzed side reactions and therefore significant solvent is required. This means that significant solvent can be saved from this process by adding concentrated base to the coupling solvent.
[011] To demonstrate the effectiveness of this new process a batch of 24 peptides were assembled using an automated peptide synthesizer modified to perform the in-situ solvent recycling step during each cycle.
[012] Materials and methods:
2a Date Recue/Date Received 2022-07-06 [013] All peptides were synthesized using a LTBERTY BLUETM PRTMETm system (CEM Corp., Matthews, NC, USA) allowing for automated in-situ solvent recycling and evaporation based washing. The peptides were synthesized at 0.05mmol scale with 10 equivalents of amino acid using CarbolVIAX1 m coupling with amino acidicarbodiimide/
ethyl 2-cyano-2-(hydroxyimino)acetate (AA/DIC/Oxyma) (1:2:1) based activation for 100 sec at 90 C E. Atherton, N. L. Benoiton, E. Brown, R. Sheppard and B. J.
Williams, "Racemization of Activated, Urethane-protected Amino-acids by p-Dimethylaminopyridine. Significance in Solid Phase Peptide Synthesis," J.C.S
(Ihem.
Comm., pp. 336-337, 1981). ProTide resins (CEM Corp.) based on TentaGele technology were used for synthesis with either a Rink Amide linker or a Cl-TCP(C1) linker with unactivated loading of the first amino acid with DIEA at 90 C for
5 min.
The deprotection step was performed for 50 sec at 95 C and initiated by adding 0.5mL
of 50% pyrrolidine directly to the coupling solution. A single 1x4mL wash was used in between the deprotection and coupling steps. Peptides were cleaved with Trifluoroacetic acid (TFA)/triisopropy1silane/water/2,2'-(ethylenedioxy)diethanethiol (TFA/TIS/H20/1DODO (92.5:2.5:2.5:2.5) for 30 min at 38 C using a RAZORTM
cleavage system (CEM Corp.).
[014]
"e =
:
The deprotection step was performed for 50 sec at 95 C and initiated by adding 0.5mL
of 50% pyrrolidine directly to the coupling solution. A single 1x4mL wash was used in between the deprotection and coupling steps. Peptides were cleaved with Trifluoroacetic acid (TFA)/triisopropy1silane/water/2,2'-(ethylenedioxy)diethanethiol (TFA/TIS/H20/1DODO (92.5:2.5:2.5:2.5) for 30 min at 38 C using a RAZORTM
cleavage system (CEM Corp.).
[014]
"e =
:
6 I
Cl-TCP(CD-ProTide RA-ProTide [015] Results and discussion:
# Peptide Disease Area Resin UPLC Synthesi ,Used purity s Time 1 GRP Regulates RA 81 1:22 VPLPAGGGTVLTKMITRGNHWAVGHTM-NH2 _Gastrin ReleaseProTide 2 Glucagon Hypoglycemia RA 75 1:28 H-IISQGTFTSDYSKYLDSRRAQDFVQWLMNT-NII2 ProTide 3 Bivalirudin C1-2-C1-71 1:05 H-fPRPGGGGNGDFEE1PEEYL-OH Blood thinner l'rt 4 Angiotensin Vasoconstrictor Cl-2-C1-82 0:30 H-NRVYVHPF-OH Trt PTH 1-34 Osteoporosis RA 70 1:43 H-SVSEIQLMHNLGKIILNSMERVEWLRKKLQDVHNF- ProTide Gonadorelin Fertility RA 91 0:35 pE HWS YGLRPG -NH2 ProTide
Cl-TCP(CD-ProTide RA-ProTide [015] Results and discussion:
# Peptide Disease Area Resin UPLC Synthesi ,Used purity s Time 1 GRP Regulates RA 81 1:22 VPLPAGGGTVLTKMITRGNHWAVGHTM-NH2 _Gastrin ReleaseProTide 2 Glucagon Hypoglycemia RA 75 1:28 H-IISQGTFTSDYSKYLDSRRAQDFVQWLMNT-NII2 ProTide 3 Bivalirudin C1-2-C1-71 1:05 H-fPRPGGGGNGDFEE1PEEYL-OH Blood thinner l'rt 4 Angiotensin Vasoconstrictor Cl-2-C1-82 0:30 H-NRVYVHPF-OH Trt PTH 1-34 Osteoporosis RA 70 1:43 H-SVSEIQLMHNLGKIILNSMERVEWLRKKLQDVHNF- ProTide Gonadorelin Fertility RA 91 0:35 pE HWS YGLRPG -NH2 ProTide
7 Triptorelin Breast Cancer, RA 73 0:35 pEHWSYwLRPG-NH2 Prostrate ProTide Cancer,
8 Liraglutide Diabetes RA 80 1:31 H-HAEGTFTSDVSSYT,ECIQ AAK(y-E- ProTide pa1mitoyDEFIAWLVRGRG-NH2 Exenatide Diabetes RA 74 1:58 H- ProTide HGEGTFTSDLSKQ1VfEEEAVRLFIEWLKNGGPSSGAPPP
MOG (35-55) Multiple RA 71 1:05 H-MEVGWYRSPFSRVVHLYRNCIK-NT-12 Sclerosis ProTide 11 Secretin Osmoregulation RA 70 1:19 II-IIDGTFTSELSRLRDSARIARLLOGLV-NII2 ProTide 12 Teriparatide Osteoporosis RA 60 1:43 H-S VSE I QLMHNLGKHLNSMERVEWLRKKLQDVHNF- ProTide 13 GLP-1 (7-37) Diabetes RA 74 1,34 H-HAEGTFTSDVSSYLEGQ1\AKEFIAWLVKGRG-NH2 ProTide 14 Magainin 1 Antibiotic RA 79 1:11 H-GIGKFLHSAGKFGKAFVGEIMKS-NH2 ProTide Tetracosactide Adrenal Cortex RA 77 1:13 H-SYSMEHFRWGKPVGKKRRPVIWYP-NH2 stimulant ProTide 16 [ArgaVasopressin Hormone (bloodRA
H-CYPQNCPRG-NH2 vessel ProTide 17 Ubiquitin Protein RA > 60 344 MQIFVKTUMKTITLEVEPSDTIENVKAICIQDKEGIPPDQ signaling agent ProTide 18 Parasin I Antibiotic RA 87 0:59 H-KGRGKQGGKVRAKAKTRSS-NH2 ProTide 19 Dynorphin A Opioid RA 71 0:53 I I-YGGFLRRI RP KLKWD NQ -NI 12 Research ProTide ACP Fatty Acid RA 92 0:32 11- V QAAID Y1NG -NH2 Synthesis ProTide 21 BAM 3200 Opioid RA 70 1:16 H-YGGFMRRVGRPEWWMDYQKRYGGFL-NH2 Research ProTide 22 1-1W-TAT (47-57) HIV/AIDS RA
93 0.31 Fmoc-YGRKERRQRRR-NH2 Research ProTide 23 HIV-TAT (48-60) HIV/AIDS RA 88 0:39 Fmoc-GRKKRRQIIRRPPQ-N112 Research ProTide 24 Pramlintide Diabetes RA 72 1:52 KCNTATCATQRLANFLVHSSNNFGPILPPTNVUSNTY-- Pro'l'ide [016] Table 1 Automated Sequential Batch Synthesis of 24 Peptides [017] All peptides synthesized in Table 1 gave the desired target as the major peak with a standard cycle time of 2 minutes and 58 seconds. The in-situ solvent recycling process allowed for 0.5mL of a concentrated pyrrolidine (BP 87 C) solution to be added to the end of the coupling step (without draining). An advantage of this setup was that the deprotection immediately proceeded very close to the desired temperature (95 C) because the coupling solution was already at 90 C. During the deprotection process a vacuum was applied and the pyrrolidine was evaporated and subsequently condensed in the waste container. This allowed only a single wash step (1 x 4mL) to be required at the end of the deprotection step.
[018] Total synthesis time for entire batch: 32.6 hours [019] This new process provided a significant reduction in standard cycle time (2 minutes 67 seconds) from (a) ¨ elimination of the coupling drain time, (b) ¨
elimination of the deprotection delivery time between steps, and (c) ¨ elimination of the temperature ramp time for the deprotection step thereby allowing a shorter deprotection time to be used. Additionally, significant solvent savings were possible with the complete elimination of the deprotection solvent during each cycle.
[020] In the drawings and specification there has been set forth a preferred embodiment of the invention, and although specific terms have been employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being defined in the claims.
MOG (35-55) Multiple RA 71 1:05 H-MEVGWYRSPFSRVVHLYRNCIK-NT-12 Sclerosis ProTide 11 Secretin Osmoregulation RA 70 1:19 II-IIDGTFTSELSRLRDSARIARLLOGLV-NII2 ProTide 12 Teriparatide Osteoporosis RA 60 1:43 H-S VSE I QLMHNLGKHLNSMERVEWLRKKLQDVHNF- ProTide 13 GLP-1 (7-37) Diabetes RA 74 1,34 H-HAEGTFTSDVSSYLEGQ1\AKEFIAWLVKGRG-NH2 ProTide 14 Magainin 1 Antibiotic RA 79 1:11 H-GIGKFLHSAGKFGKAFVGEIMKS-NH2 ProTide Tetracosactide Adrenal Cortex RA 77 1:13 H-SYSMEHFRWGKPVGKKRRPVIWYP-NH2 stimulant ProTide 16 [ArgaVasopressin Hormone (bloodRA
H-CYPQNCPRG-NH2 vessel ProTide 17 Ubiquitin Protein RA > 60 344 MQIFVKTUMKTITLEVEPSDTIENVKAICIQDKEGIPPDQ signaling agent ProTide 18 Parasin I Antibiotic RA 87 0:59 H-KGRGKQGGKVRAKAKTRSS-NH2 ProTide 19 Dynorphin A Opioid RA 71 0:53 I I-YGGFLRRI RP KLKWD NQ -NI 12 Research ProTide ACP Fatty Acid RA 92 0:32 11- V QAAID Y1NG -NH2 Synthesis ProTide 21 BAM 3200 Opioid RA 70 1:16 H-YGGFMRRVGRPEWWMDYQKRYGGFL-NH2 Research ProTide 22 1-1W-TAT (47-57) HIV/AIDS RA
93 0.31 Fmoc-YGRKERRQRRR-NH2 Research ProTide 23 HIV-TAT (48-60) HIV/AIDS RA 88 0:39 Fmoc-GRKKRRQIIRRPPQ-N112 Research ProTide 24 Pramlintide Diabetes RA 72 1:52 KCNTATCATQRLANFLVHSSNNFGPILPPTNVUSNTY-- Pro'l'ide [016] Table 1 Automated Sequential Batch Synthesis of 24 Peptides [017] All peptides synthesized in Table 1 gave the desired target as the major peak with a standard cycle time of 2 minutes and 58 seconds. The in-situ solvent recycling process allowed for 0.5mL of a concentrated pyrrolidine (BP 87 C) solution to be added to the end of the coupling step (without draining). An advantage of this setup was that the deprotection immediately proceeded very close to the desired temperature (95 C) because the coupling solution was already at 90 C. During the deprotection process a vacuum was applied and the pyrrolidine was evaporated and subsequently condensed in the waste container. This allowed only a single wash step (1 x 4mL) to be required at the end of the deprotection step.
[018] Total synthesis time for entire batch: 32.6 hours [019] This new process provided a significant reduction in standard cycle time (2 minutes 67 seconds) from (a) ¨ elimination of the coupling drain time, (b) ¨
elimination of the deprotection delivery time between steps, and (c) ¨ elimination of the temperature ramp time for the deprotection step thereby allowing a shorter deprotection time to be used. Additionally, significant solvent savings were possible with the complete elimination of the deprotection solvent during each cycle.
[020] In the drawings and specification there has been set forth a preferred embodiment of the invention, and although specific terms have been employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being defined in the claims.
Claims (8)
1. A method of deprotection in batch-wise solid phase peptide synthesis comprising:
adding a base deprotection composition to a mixture of a coupling solution, a growing peptide chain, and any excess activated amino acid from a preceding coupling step;
wherein the deprotection composition is added to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling step in an amount that is less than 1/3 of the volume of the coupling solution;
without any draining step between the coupling step of the preceding coupling step and the addition of the deprotection composition for a successive cycle;
and with the coupling solution at a temperature of at least 30 C.
adding a base deprotection composition to a mixture of a coupling solution, a growing peptide chain, and any excess activated amino acid from a preceding coupling step;
wherein the deprotection composition is added to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling step in an amount that is less than 1/3 of the volume of the coupling solution;
without any draining step between the coupling step of the preceding coupling step and the addition of the deprotection composition for a successive cycle;
and with the coupling solution at a temperature of at least 30 C.
2. A method according to claim 1 wherein the deprotection composition is an organic base.
3. A method according to claim 1 or 2 using Fmoc solid phase peptide chemistry.
4. A method according to any one of claims 1 to 3 wherein the deprotection composition has a concentration of at least 50% base by volume.
5. A method of deprotection in batch-wise solid phase peptide synthesis comprising:
adding a base deprotection composition to a mixture of a coupling solution, a growing peptide chain, and any excess activated amino acid from a preceding coupling cycle step;
wherein the deprotection composition is added to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling cycle step in an amount that is less than 1/3 of the volume of the coupling solution;
without any draining step between the coupling step of the preceding coupling cycle step and the addition of the deprotection composition for a successive cycle which Date Reçue/Date Received 2022-07-06 removes at least 50% of the volume of the preceding coupling cycle step coupling solution; and with the coupling solution at a temperature of at least 30 C.
adding a base deprotection composition to a mixture of a coupling solution, a growing peptide chain, and any excess activated amino acid from a preceding coupling cycle step;
wherein the deprotection composition is added to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino acid from the preceding coupling cycle step in an amount that is less than 1/3 of the volume of the coupling solution;
without any draining step between the coupling step of the preceding coupling cycle step and the addition of the deprotection composition for a successive cycle which Date Reçue/Date Received 2022-07-06 removes at least 50% of the volume of the preceding coupling cycle step coupling solution; and with the coupling solution at a temperature of at least 30 C.
6. A method according to claim 5 wherein the deprotection composition is an organic base.
7. A method according to claim 5 or 6 using Fmoc solid phase peptide chemistry.
8. A method according to any one of claims 5 to 7 wherein the deprotection composition concentration is at least 50% base by volume.
Date Recue/Date Received 2022-07-06
Date Recue/Date Received 2022-07-06
Applications Claiming Priority (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201662383397P | 2016-09-03 | 2016-09-03 | |
| US62/383,397 | 2016-09-03 | ||
| US15/299,931 | 2016-10-21 | ||
| US15/299,931 US10125163B2 (en) | 2015-10-23 | 2016-10-21 | Solid phase peptide synthesis |
| USPCT/US2016/058181 | 2016-10-21 | ||
| PCT/US2016/058181 WO2017070512A1 (en) | 2015-10-23 | 2016-10-21 | Improvements in solid phase peptide synthesis |
| US15/490,090 US10239914B2 (en) | 2015-10-23 | 2017-04-18 | In-situ solvent recycling process for solid phase peptide synthesis at elevated temperatures |
| US15/490,090 | 2017-04-18 | ||
| PCT/US2017/028254 WO2018044356A1 (en) | 2016-09-03 | 2017-04-19 | In-situ solvent recycling process for solid phase peptide synthesis at elevated temperatures |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA3034810A1 CA3034810A1 (en) | 2018-03-08 |
| CA3034810C true CA3034810C (en) | 2023-09-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA3034810A Active CA3034810C (en) | 2016-09-03 | 2017-04-19 | In-situ solvent recycling process for solid phase peptide synthesis at elevated temperatures |
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| Country | Link |
|---|---|
| EP (1) | EP3507299B1 (en) |
| JP (1) | JP2019530659A (en) |
| CN (1) | CN109715647A (en) |
| CA (1) | CA3034810C (en) |
| DK (1) | DK3507299T3 (en) |
| WO (1) | WO2018044356A1 (en) |
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| US7902488B2 (en) * | 2003-06-23 | 2011-03-08 | Cem Corporation | Microwave-assisted peptide synthesis |
| US7803351B2 (en) * | 2004-08-20 | 2010-09-28 | Washington University | Blood brain barrier permeation peptides |
| US8314208B2 (en) * | 2006-02-10 | 2012-11-20 | Cem Corporation | Microwave enhanced N-fmoc deprotection in peptide synthesis |
| CA2808270A1 (en) * | 2010-08-16 | 2012-02-23 | Cem Corporation | Water soluble solid phase peptide synthesis |
| US8906681B2 (en) * | 2011-08-02 | 2014-12-09 | The Scripps Research Institute | Reliable stabilization of N-linked polypeptide native states with enhanced aromatic sequons located in polypeptide tight turns |
| EP2703073A1 (en) * | 2012-08-31 | 2014-03-05 | Biotage AB | Apparatus and method for solid phase synthesis |
| US9169287B2 (en) * | 2013-03-15 | 2015-10-27 | Massachusetts Institute Of Technology | Solid phase peptide synthesis processes and associated systems |
| EP3365352A4 (en) * | 2015-10-23 | 2019-06-05 | CEM Corporation | Improvements in solid phase peptide synthesis |
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| CN109715647A (en) | 2019-05-03 |
| EP3507299B1 (en) | 2022-07-27 |
| DK3507299T3 (en) | 2022-09-05 |
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